CYCLOTIDES AS IMMUNOSUPPRESSIVE AGENTS
20170239327 · 2017-08-24
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07K14/00
CHEMISTRY; METALLURGY
International classification
A61K38/16
HUMAN NECESSITIES
Abstract
The present invention relates to a pharmaceutical composition comprising a cyclotide for use in immune suppression as well as to a method for immune suppression comprising the step of administering an effective amount of a pharmaceutical composition comprising such a cyclotide to a subject in need thereof. The present invention also relates to a pharmaceutical composition comprising a cyclotide for use in treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. Likewise, the present invention also relates to a method for treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. The present invention further relates to a method of screening for and/or selecting an immunosuppressive cyclotide or a mutation which results in a mutated cyclotide having an induced or enhanced immunosuppressive activity. The present invention further relates to a method of producing an immunosuppressive cyclotide or an immunosuppressive pharmaceutical composition. The present invention further relates to a mutated cyclotide having immunosuppressive activity and a pharmaceutical composition comprising the same.
Claims
1-35. (canceled)
36. A method for immunosuppression, the method comprising administering an effective amount of a non-grafted cyclotide to a subject in need thereof, wherein said cyclotide comprises a head-to tail cyclized peptide, wherein said head-to tail cyclized peptide comprises six conserved cysteine residues capable to form three disulfide bonds arranged in a cyclic cystine-knot (CCK) motif.
37. The method of claim 36, wherein said cyclotide does not include another therapeutic peptide.
38. The method of claim 36, wherein said cyclotide comprises an amino acid sequence of formula II; wherein formula II comprises: TABLE-US-00009 (SEQ ID NO. 17) Xxx.sub.1-Leu-Pro-Val-Cys-Gly-Glu-Xxx.sub.2-Cys-Xxx.sub.3-Gly- Gly-Thr-Cys-Asn-Thr-Pro-Xxx.sub.1-Cys-Xxx.sub.1-Cys-Xxx.sub.1- Trp-Pro-Xxx.sub.1-Cys-Thr-Arg-Xxx.sub.1,; wherein Xxx.sub.1 comprises any amino acid, non-natural amino acid or peptidomimetic; wherein Xxx.sub.2 comprises any amino acid, non-natural amino acid or peptidomimetic but not Lys; and wherein Xxx.sub.3 comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys.
39. The method of claim 36, wherein said cyclotide comprises a cyclic backbone having the structure of formula I, wherein formula I comprises the amino acid sequence:
Cyclo(C[X.sub.1 . . . Xa]C[XI1 . . . XIb]C[XII1 . . . XIIc]C[XIII1 . . . XIIId]C[XIV1 . . . XIVe]C[XV1 . . . XVf]); wherein C is cysteine; wherein each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe], and [XV1 . . . XVf] represents one or more amino acid residues, wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e, and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 20.
40. The method of claim 36, wherein said cyclotide has an anti-proliferative effect on (an) immune cell(s) and/or suppresses/reduces the effector function(s) of (an) immune cell(s).
41. The method of claim 39, wherein a is 3 to 6, b is 4 to 8, c is 3 to 10, d is 1, e is 4 to 8, and/or f is 5 to 13.
42. The method of claim 36, wherein said cyclotide comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (ii) an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 12, 15, and 16; (iii) an amino acid sequence encoded by a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and; or (iv) an amino acid sequence that is at least 90% identical to any amino acid sequence of (i) to (iii).
43. The method of claim 36, wherein said cyclotide comprises kalata B1 or kalata B2.
44. The method of claim 36, wherein said cyclotide is administered so that cytostatic but no cytotoxic activity occurs.
45. The method of claim 36, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 1 to 50 μM, preferably in the range of 3 to 10 μM, more preferably 4 to 9 μM, and even more preferably in the range of 5 to 9 μM.
46. The method of claim 36, wherein said pharmaceutical composition further comprises one or more additional immunosuppressants.
47. The method of claim 46, wherein said additional immunosuppressant comprises Cyclosporine A, Muromonab-CD3 or Basiliximab.
48. The method of claim 36, for the treatment of a subject suffering from a disorder selected from the group consisting of an autoimmune disorder, a hypersensitivity disorder, and a lymphocyte-mediated inflammation.
49. The method of claim 48, wherein said autoimmune disorder is selected from the group consisting of Multiple Sclerosis, Psoriasis, Systemic Lupus Erythematosus, Sjögren's syndrome, Rheumatoid Arthritis, Idiopathic Thrombocytopenic Purpura, Diabetes, Vasculitis, and Crohn's disease.
50. The method of claim 48, wherein said lymphocyte-mediated inflammation comprises a T cell-mediated inflammation.
51. The method of claim 48, wherein said lymphocyte-mediated inflammation comprises keratoconjunctivitis sicca or dry eye syndrome (DES).
52. The method of claim 36, wherein the amino acid sequence of said cyclotide is radio-labelled, fluorescence-labelled or biotin-labelled.
53. The method of claim 36, wherein: the proliferation of (an) immune cell(s); the effector function(s) of (an) immune cell(s); the degranulation/cytotoxicity of (an) immune cell(s); the expression of a cytokine surface receptor on (an) immune cell(s); the proliferation of (primary) activated lymphocytes; the proliferation of peripheral blood mononuclear cells (PBMC); secretion/production of IL-2, IFN-gamma and/or TNF-alpha; degranulation/cytotoxicity of CD107a+CD8+ PBMCs; and/or expression of IL-2 surface receptor CD25 is/are suppressed.
54. The method of claim 36, wherein said cyclotide suppresses/reduces secretion/production of IL-2, IFN-gamma and/or TNF-alpha; suppresses/reduces degranulation/cytotoxicity of CD107a+CD8+ PBMCs; and/or suppresses/reduces expression of IL-2 surface receptor CD25.
55. The method of claim 36, wherein the anti-proliferative effect or suppression/reduction is mediated in an IL-2-, IFN-gamma- and/or TNF-alpha-depending manner and/or can be antagonized by IL-2.
56. The method of claim 36 comprising oral administration.
57. A mutated cyclotide having immunosuppressive activity, said cyclotide comprising an amino acid sequence that is at least 90% identical to an amino acid selected from the group consisting of SEQ ID NOs: 1, 2, and 3.
58. Method of screening for and/or selecting an immunosuppressive cyclotide, the method comprising: (i) contacting a cyclotide or a plant extract containing a cyclotide with an activated immune cell and determining the proliferative activity of said cell, wherein a suppressed or reduced proliferative activity as compared to a control is indicative for the immunosuppressive activity of the cyclotide; or (ii) administering to an animal model a pharmaceutically effective amount of a cyclotide or a plant extract containing a cyclotide and determining one or more parameters of the immune system, wherein the suppression or reduction of one or more parameters of the immune system as compared to a control is indicative for the immunosuppressive activity of the cyclotide.
Description
[0214] The Figures show:
[0215]
[0216]
[0217]
[0218]
[0219]
[0220]
[0221]
[0222]
[0223]
[0224]
[0225]
[0226]
[0227] B. IL2 release in human T-cells after treatment with cyclotide. Human T-cells (provided by A. Dohnal, PhD; from CCRI, Vienna) 4×106/mL were seeded in 96-well flat-bottom plates (100 μL/well) and Incubated for two hours at 37° C. before they were stimulated with CsA (5 mg/mL), T20K (4 μM) and V10K (4 μM). After another two hours PHA-L (10 μg/mL) was added to the appropriate wells and incubated over night at 37° C. On the next day T-cells were re-stimulated with lonomycin (500 ng/mL) and PMA (50 ng/mL) for 6 hours at 37° C. Cells were then centrifuged at 3000 rpm for 5 minutes to gain their supernatants. Supernatants of stimulated T-cells were analyzed for their IL2 release using a human IL-2 ELISA Kit from eBIoscience according to the manufacturer's instructions. The color reaction was evaluated at an optical density of 450 nm by the microplate reader Synergy H4 (BioTek). PHA-L stimulation of human T-cells illustrated highest IL2 release, also V10K and PMA+lonomycin stimulation achieved comparable results, whereas untreated and CsA treated cells showed no production of this cytokine. In addition, T-cells incubated with the cyclotide T20K demonstrated a significant inhibition of cell proliferation in accordance to the IL2 level. C. IL-2 gene expression analysis using RT-PCR. Total cellular RNA was isolated from PHA-L-activated cells that were incubated with medium, CsA or T20K for 4 hours. RT-PCR was carried out using specific primers for indicated gene. The data were normalized to the Ct value of the internal housekeeping gene 18s rRNA and the relative mRNA level in the untreated stimulated group was used as calibrator. Data were expressed as mean+SD of three independent experiments.
[0228]
[0229]
[0230]
[0231]
[0232]
[0233]
[0234]
[0235]
[0236]
[0237]
[0238]
[0239]
[0240] The Examples Illustrate the Invention.
EXAMPLE 1
Material and Methods
[0241] Extraction Preparation and Purification of Plant Cyclotides.
[0242] Oldenlandia affinis (R&S) DC. plants were grown in the glass house at the Department of Pharmacognosy (University of Vienna) from seeds that were obtained as a gift from David Craik (Institute for Molecular Biosciences, University of Queensland). Aerial parts of the plants have been harvested and dried. Plant material was pulverized using a rotor grinder and extracted twice overnight in dichloromethane:methanol (1:1 v/v). The extracts were concentrated on a roto-evaporator and were lyophilized. The dried extracts were dissolved in solvent A (ddH.sub.2O with 0.1% TFA) and in-batch pre-purified with C.sub.18 solid phase extraction (ZEOprep 60 Å, C.sub.18 irregular 40-63 μm; ZEOCHEM, Uetikon, Switzerland). To separate the hydrophilic non-cyclotide compounds from the hydrophobic cyclotide compounds, the C.sub.18-beads were washed with 10% solvent B (90% acetonitrile in ddH.sub.2O with 0.08% TFA) and eluted with 80% solvent B. The eluate containing cyclotides was analyzed by MALDI-TOF MS and reconstituted in ddH.sub.2O at 10 mg/mL for biological assays or used for nano LC-MS/MS analysis and further purification. Kalata B1 was purified from crude O. affinis extract by HPLC using a Perkin Elmer Series 200 system with preparative (Phenomenex Jupiter, 10 μm, 300 Å, 250×21.2 mm; 8 mL/min) and semi-preparative (Kromasil C.sub.18, 5 μm, 100 Å, 250×10 mm; 3 mL/min) RP-C.sub.18 HPLC columns and linear gradients from 0-80% solvent B in 80 min. Eluting peptides were monitored with UV-absorbance (A.sub.280), collected manually and lyophilized. Purity and quality of kalata B1 was assessed by analytical HPLC and MALDI-TOF MS.
[0243] Nano LC-MS and LC-MS/MS Analysis.
[0244] Crude, ZipTip™ prepared or digested plant extracts (C.sub.18 pre-purified O. affinis extract, see above) were analyzed by nano LC-MS or LC-MS/MS on an Ultimate 3000 nano HPLC system controlled by Chromeleon 6.8 software (Dionex, Amsterdam, The Netherlands). For LC analysis, samples of O. affinis extract (1-5 μL) were injected, pre-concentrated using Dionex PepMap™ C.sub.18 cartridges (300 μm×5 mm, 5 μm, 100 Å) and separated by nano-RP-HPLC prior to online MS analysis using a Dionex Acclaim PepMap™ C.sub.18 column (150 mm×75 μm, 3 μm, 100 Å; 300 nL/min). The mobile phase consisted of solvent C (0.1% aqueous formic acid) and solvent D (90/10 acetonitrile/0.08% aqueous formic acid). Peptides were eluted using a linear gradient of 4-90% D in 35 min, 5-min hold at 90% D, followed by a return to 4% D for a 20-min equilibration. For LC-MS/MS analysis aliquots (1-10 μL) of tryptic or endo-GluC digested plant extracts were pre-concentrated and separated by C.sub.18 nano LC as described above, using several LC gradients of up to 120 min duration (e.g., 4-60% B in 100 min, 60-90% B in 1 min and finally a 5-min hold at 90% B, followed by a return to 4% B for a 10-min equilibration). Eluated peptides were directly introduced into the nanospray source. Mass spectrometry experiments were performed on a hybrid quadrupole/Inear ion trap 4000 QTRAP MS/MS system (ABSciex, Foster City, Calif., USA) running with the Analyst 1.5.1 software package. The 4000 QTRAP equipped with a nano-spray source was operated in positive ionization mode. LC-MS analyses for cyclotide quantification and identification by molecular weight were performed using Enhanced Multiple Scan (EMS) acquisition with a scan speed of 1000 amu/sec in the mass range from 400-1400 Da. LC-MS data were analyzed by “LC-MS reconstruct” in the MW range from 2700-3500 Da and by using several signal-to-noise filter settings to obtain the molecular weight and validity score of all peptide peaks. LC-MS/MS analyses were performed using information Dependent Acquisition (IDA). The acquisition protocol used to provide mass spectral data for database searching involved the following procedure: mass profiling of the HPLC eluant using EMS; ions over the background threshold were subjected to examination using the Enhanced Resolution (ER) scan to confirm charge states of the multiply charged molecular ions. The most and next most abundant ions in each of these scans with a charge state of +2 to +4 or with unknown charge were subjected to CID using rolling collision energy. Enhanced product ion scan was used to collate fragment ions and present the product ion spectrum for subsequent database searches.
[0245] Enzymatic Digest and Peptide Sequencing Using Database Analysis.
[0246] C.sub.18 prepurified O. affinis extract cyclotides were prepared for MS/MS sequencing as described earlier (Chen, 2005, J Biol Chem, 280, 22395-22405; Ireland, 2006, Biochem J, 400, 1-12). The extract was reduced, alkylated with iodoacetamide and enzymatic digested using trypsin or endo-GluC (Sigma-Aldrich, Austria). Digested peptide extracts were analyzed with nano LC-MS/MS as described above and IDA data were used for further analysis. Database searching of LC-MS/MS data was carried out using the ProteinPilot™ software and the Paragon algorithm with the custom-made ERA database tool for the Identification of cyclotides (Colgrave, 2010, Biopolymers, 94, 592-601).
[0247] Relative Quantification of Cyclotides Using Nano LC-MS Analysis.
[0248] C.sub.18 prepurified O. affinis extract was separated by one dimensional nano LC-MS as described above. Cyclotide peaks were quantified by relative area under curve (all peaks at 214 nm absorbance from 15-55 min were processed) using the quantification wizard of Chromeleon 6.8 software. Peaks in the LC chromatogram were identified by molecular weight and retention time from corresponding LC-MS peaks. Quantification was performed on five independent LC-MS experiments and relative cyclotide abundance is presented as mean±SEM.
[0249] Preparation of Human Peripheral Blood Mononuclear Cells and Cell Culture.
[0250] Human peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adult donors obtained from the Blood Transfusion Centre (University Medical Center, Freiburg, Germany). Venous blood was centrifuged on a LymphoPrep™ gradient (density: 1.077 g/cm.sup.3, 20 min, 500×g, 20° C.; Progen, Heidelberg, Germany). Afterwards cells were washed twice with medium and cell viability and concentration was determined using the trypan blue exclusion test. PBMC were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (PAA, Coelbe, Germany), 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin (all from Invitrogen, Karlsruhe, Germany). The cells were cultured at 37° C. in a humidified incubator with a 5% CO.sub.2/95% air atmosphere. All experiments conducted on human material were approved by the Ethics committee of the University of Freiburg.
[0251] Alternative Purification of Human Peripheral Mononuclear Cells (PBMCs).
[0252] PBMCs were isolated from blood samples of healthy adults that were provided by the transfusion center of the university hospital in Freiburg (Germany). Venous blood was diluted 1:2 (v/v) with PBS and centrifuged with a LymphoPrep-gradient (using 15 ml diluted blood and 20 ml LymphoPrep solution); density: 1.077 g/cm.sup.3, 20 min, 500×g, 20° C.). The lymphocyte-enriched layer was transferred into a new vessel and washed three times with PBS and centrifuged again (10 min, twice with 300×g und last time with 800 rpm, 20° C.). For the following experiments, the cells were either stained with CFSE or diluted with medium to 4*10.sup.6 cells/ml. Cells were counted in alight microscope using trypan blue staining and a hemocytometer.
[0253] Activation and Treatment of PBMCs.
[0254] PBMCs (10.sup.5) were stimulated with anti-human CD3 (clone OKT3) and anti-human CD28 (clone 28.2) mAbs (both from eBioscience, Frankfurt, Germany) for 72 hrs in the presence of medium, the control agents camptothecin (CPT; 30 μg/mL: Tocris, Eching, Germany) and Triton-X 100 (0.5%; Carl Roth, Karlsruhe, Germany) or different concentrations of O. affinis extract, melittin (PolyPeptide, Strasbourg, France) or kalata B1, respectively. After cultivation, the cells were assessed in bioassays as described in the text.
[0255] Alternative Activation and Treatment of PBMCs.
[0256] Following purification, PBMCs were equilibrated for 2 h at 37° C. Afterwards 100 μl PBMCs (4*10.sup.6 cells/ml) were preincubated in a 96-well plate for 2 h with CsA (cyclosporine A) or cyclotides, transferred to a new plate and stimulated with 10 μg/ml PHA-L for 1 h. This was followed by washing of each well with 100 μl PBS (centrifugation for 5 min, 1000 rpm, 20° C.) and re-suspending the cells in 100 μl medium for further assays.
[0257] Determination of Cell Proliferation and Cell Division.
[0258] For cell proliferation and cell division tracking analysis PBMC were harvested and washed twice in cold PBS and resuspended in PBS at a concentration of 5×10.sup.6 cells/mL. Cells were incubated for 10 min at 37° C. with carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 μM: Sigma-Aldrich, Taufkirchen, Germany). The staining reaction was stopped by washing twice with complete medium. Afterwards, the cell division progress was analysed using flow cytometric analysis.
[0259] Alternative Analysis of Cell Proliferation and Cell Division Using CFSE Staining.
[0260] Purified PBMCs (5*106 cells/ml) were incubated with 0.5 mM of the fluorescent dye CFSE (5-carbofluoreszenein-ciacetat-succinylester) for 10 min at 37° C. The reaction was stopped using medium, cells were washed one time with medium by centrifugation (10 min, 300×g, 20° C.) and diluted with medium to 4*10.sup.6 cells/ml.
[0261] Determination of PBMC Apoptosis and Necrosis Using Annexin V and Propidium Iodide Staining.
[0262] The levels of apoptosis were determined using the annexin V-FITC apoptosis detection kit (eBioscience, Frankfurt, Germany) according to the manufacturer's instructions. After annexin V staining, propidium iodide solution (PI; eBioscience) was added and the cells were incubated in the dark, followed by a flow cytometric analysis to determine the amount of apoptosis and necrosis. CPT (30 μg/mL) and Triton-X 100 (0.5%) was used as positive controls for apoptosis and necrosis, respectively.
[0263] Mice. C57BL/6 mice (10-16 weeks old) were bred and maintained in the Monash University Animal Services facilities. All experiments were conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes (NHMRC, 1997), after approval by the Monash University Animal Ethics committee (Clayton/Melbourne, Australia).
[0264] Induction and Clinical Assessment of EAE.
[0265] A total of 200 μg of the encephalitogenic peptide MOG.sub.35-55 (MEVGWYRSPFSRWHLYRNGK; GL Biochem, Shanghai, China) emulsified in CFA (Sigma) supplemented with 4 mg/ml Mycobacterium tuberculosis (BD) was injected subcutaneously into the flanks. Mice were then immediately injected intravenously with 350 ng of pertussis vaccine (List Biological Laboratories, Campbell, U.S.A.) and again 48 hr later (Bernard J Mol Med 75, 1997, 77-88; Albouz-Abo Eur J Biochem 246, 1997, 59-70; Hvas Scand J Immunol 46, 1997, 195-203; Johns Mol Immunol 34, 1997, 33-38; Menon J Neurochem 69, 1997, 214-222). Animals were monitored daily and neurological impairment was quantified on an arbitrary clinical scale: 0, no detectable impairment; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and ascending paralysis; 5, moribund or deceased (Liu Nat Med 4, 78-83 1998; Slavin Autoimmunity 28, 109-120 1998). Under recommendation of the animal ethics committee, mice were euthanised after reaching a clinical score of 4.
[0266] Antibodies and Recombinant Proteins.
[0267] The mouse anti-MOG mAb (clone 8-18C5) was purified from hybridoma culture supernatants on Protein G-Sepharose 4 Fast Flow column (GE Healthcare) according to the manufacturer's instructions. Antiserum to MOG.sub.35-55 peptide (Ichikawa Int Immunol 8, 1996, 1667-1674; Ichikawa J Immunol 157, 1996, 919-926) was raised in rabbits by procedures similar to those described previously (Bernard Clin Exp Immunol 52, 1983, 98-106; Pedersen J Neuroimmunol 5, 1983, 251-259). The extracellular domain of mouse MOG (amino acid residues 1-117 of the mature protein) (rMOG) was produced in the E. coli strain M15pREP4 using the pQE9 expression vector (Qiagen, Australia) to incorporate an amino-terminal histidine tag as per manufacturer's instructions. A clarified bacterial lysate containing rMOG was loaded onto a Ni-NTA Superflow (Qiagen, Australia) column under denaturing conditions (6 M Guanidine-HCl, 100 mM NaH.sub.2PO.sub.4, 10 mM Tris pH 8.0,) as per the manufacturer's instructions using a BioLogic LP Chromatography System (Bio-Rad Laboratories, Australia). Bound protein was washed sequentially with Buffer A (8M Urea 100 mM NaH.sub.2PO.sub.4, 10 mM Tris pH 8.0), Buffer A (at pH6.3), 10 mM Tris pH 8/60% iso-propanol (to remove endotoxin) and again with Buffer A. Refolding of the bound protein was carried out by applying a linear gradient of Buffer A containing 14 mM 2-mercaptoethanol (100%-0%) vs. Buffer B (100 mM NaH.sub.2PO.sub.4, 10 mM Tris pH 8.0, 2 mM reduced glutathione, 0.2 mM oxidised glutathione) (0%-100%). This was followed by a second linear gradient of Buffer B (100%-0%) vs. Buffer C (100 mM NaH.sub.2PO.sub.4, 10 mM Tris pH 8.0) (0%-100%). The bound protein was eluted using Buffer C containing 300 mM Imidazole, then extensively dialysed against 50 mM NaCl/10 mM Tris pH 8. Protein concentration and purity were estimated using a Micro BCA assay (Bio-Rad Laboratories, Australia) and SDS-PAGE, respectively. The protein produced was varified as rMOG by western blot analysis using antibodies specific for native MOG. Endotoxin levels were determined using a Limulus Amebocyte Lysate assay (Associates of Cape Cod, Falmouth, Mass.).
[0268] Vaccination with MOG Peptide.
[0269] 200 μg of the MOG peptide were emulsified with an equal volume of IFA (Difco) and injected subcutaneously in the upper flanks (100 μl divided equally) three weeks prior to the encephalitogenic challenge. This was followed by two more injections at weekly intervals (200 g/IFA/100 μl).
[0270] Histopathology and Assessment of Inflammation, Demyelination and Axonal Damage.
[0271] At the completion of the experiments, mice were anesthetized, their blood collected (for subsequent antibody determination) and brain and spinal cord carefully removed, prior to immersion in a 4% paraformaldehyde, 0.1 M phosphate buffer solution. Segments of brain, cerebellum and spinal cord were embedded in paraffin. Sections were stained with haemotoxylin-eosin, Luxol fast blue and Bielshowsky for evidence of inflammation, demyelination and axonal damage, respectively (McQualter 2001 J Exp Med. October 1; 194(7), 873-82). Semiquantitative histological evaluation for inflammation and demyelination was performed and scored in a blind fashion as follows: 0, no inflammation; 1, cellular infiltrate only in the perivascular areas and meninges; 2, mild cellular infiltrate in parenchyma; 3, moderate cellular infiltrate in parenchyma; and 4, severe cellular infiltrate in parenchyma (Bettadapura J Neurochem 70, 199, 1593-1599 8; Okuda J Neuroimmunol 131, 2002, 115-125).
[0272] MOG-Specific Antibody Determination.
[0273] Antibody activity to rMOG and MOG.sub.35-55 in mouse sera was measured by ELISA, as previously described Ichikawa Cell Immunol 191, 1999, 97-104). Briefly, serum was collected at the end of the experiments and tested by ELISA with rMOG and MOG.sub.35-55 peptide-coated plates (Maxisorp, Nunc).
[0274] T Cell Proliferation and Cytokine Production.
[0275] Spleens were taken from mice sacrificed 32-46 days after MOG.sub.35-55 immunization. Cells were gently dispersed through a 70 μm nylon mesh (BD) into a single cell suspension, washed and cultured at 2.5×10.sup.6 cells/ml in complete RPMI (RPMI 1640 containing 10% heat-inactivated fetal calf serum (Sigma), 2 mM L-glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 50 μm 2-mercaptoethanol and 1 mm sodium pyruvate. Two hundred microliters of cell suspensions were then added to 96 well microtitre plates either alone, with MOG.sub.35-55 (20 μg/ml) or anti-CD3 and anti-CD 28 (20 μg/ml each) and incubated for 66 h at 37° C. with 5% CO.sub.2. Ten microlitres of [.sup.3H]thymidine (1 μCi/well; Amersham, Australia; diluted 1/10 in media) were added to each well for the last 18 h. Plates were harvested onto glass fibre filters and a drop of Microscint Scintillant (Perkin Elmer) was added to each well. Counts were read using a Top Count NXT Scintillation Counter (Perkin Elmer). Presented values are the mean of three wells. For cytokine assays, 2 ml of cells (5×10.sup.8 cells/ml) from spleens isolated 32-46 days after immunization were added to 24 well plates either alone or with MOG.sub.35-55 (10 gig/ml) or with anti-CD3s and anti-CD 28 (20 μg/ml each). Supernatants were collected at 48 and 72 h. Quantitation of mouse cytokine content incorporating Th1, Th2 cytokines and chemokines (IFNγ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, GM-CSF, KC, MCP-1, MIG, and TNF) were simultaneously determined using a multiplexed bead assay (Cytometric Bead Array Flex sets [CBA]) according to the manufacturer's recommended protocol (Becton Dickinson). Acquisition of 4500 events was performed using a FACScanto II flow cytometer (Becton Dickinson, San Jose, USA) and Diva software and data analysed and fitted to a 4-parameter logistic equation using the FCAP array software (Soft Flow, Pécs, Hungary). Minimum detection levels of each cytokine were: IFNγ, 5.2 μg/ml; IL-2, 1.5 μg/ml; IL-3, 4.2 μg/ml; IL-4, 0.8 μg/ml; IL-5, 4.8 μg/ml; IL-6, 6.5 μg/ml; IL-9, 10.5 μg/ml; IL-10, 16.4 μg/ml; IL-12p70, 9.2 μg/ml; IL-13, 7.3 μg/ml; GM-CSF, 9.9 μg/ml; KC, 16.2 μg/ml; MCP-1, 29 μg/ml; MIG, 11.4 μg/ml and TNF, 17.1 μg/ml.
[0276] IL-2 Surface Receptor Analysis.
[0277] Activated cells were transferred into a 96-well plate, centrifuged (5 min, 1000 rpm, 20° C.), washed one time with 100 μl FACS-buffer and stained with CD25 PE for 15 min at 4° C. Then cells were washed twice with FCS-buffer and resuspended in 100 μl FACS-buffer, transferred into FACS vials with a total volume of 250 μl and the expression of IL2 surface receptor CD25 was measured by FACS analysis using a FACSCalibur instrument (BD Biosciences).
[0278] Determination of Cytokine Release Using ELISA.
[0279] Activated cells were resuspended in 50 μl of medium, transferred into a 96-well plate and treated with 10 μg/ml PHA-L. After incubation for 24 h cells were re-stimulated with PMA (50 ng/ml) und ionomycin (500 ng/ml) for 6 h. Next, the cells were transferred into Eppendorf tubes, centrifuged (5 min, 3000 RPM, 20° C.) und 50 μl of the supernatant was again transferred into new tubes and stored at −20° C. Production of cytokines was measured and quantified using the FlowCytomix™ kit according to manufacturer's instructions.
[0280] CD107a—Degranulation Analysis
[0281] Activated cells were grown for 36 h and then treated for re-stimulation with PMA (50 ng/ml) und lonomycin (500 ng/ml) and stained with CD107a PE. After 1 h the reaction was stopped with 2 μl Golgi-Stop (1:10) and incubated for 2.5 h at 37° C. The cells were transferred into a 96-well plate, centrifuged (5 min, 1000 RPM, 20° C.) and washed with 100 μl FACS-buffer. Afterwards, PBMCs were stained with CD8 PE-Cy5 for 15 min at 4° C. and following to wash cycles with FACS-buffer the cells were resuspended in 100 μl, transferred into FACS vials with a total volume of 250 μl and the degranulation was measured by FACS analysis.
[0282] Intracellular Production of IFN-Gamma and TNF-Alpha.
[0283] Activated cells were grown for 36 h and then treated for re-stimulation with PMA (50 ng/ml), lonomycin (500 ng/ml) and brefeldin A for 6 h at 37° C. After transferring the cells into a 96-well plate, they were centrifuged (5 min, 1000 RPM, 20° C.) and washed with 100 μl FACS-buffer. Afterwards, PBMCs were stained with CD8 PE-Cy5 for 15 min at 4° C. and washed again twice with FACS-buffer. The cells were treated with 50 μl of 4% paraformaldehyde for 10 min at 4° C., washed twice with 100 μl FACS-buffer and then permeabilized by incubation with 100 μl Perm/Wash solution (1:10) for 15 min at 4° C. After centrifugation (5 min, 1000 rpm, 20° C.), PBMCs were incubated with IFN-gamma PE or TNF-alpha PE, respectively, for 30 min at 4° C. Free antibodies were washed away with Perm/Wash and PBMCs were re-suspended in 100 μl FACS-buffer. Production of IFN-gamma and TNF-alpha was individually determined by FACS analysis.
[0284] Total RNA Extraction and Reverse Transcription.
[0285] Total RNA was extracted from controls or treated cells (2×10.sup.6) frozen at −80° C. RNA-purification was performed according to the manufacturer's instructions for the RNeasy mini and Rnase-Free Dnase Set digestion kits (Qiagen, Hilden, Germany). The quantity and purity of extracted RNA was measured by spectrophotometry (Nanodrop, Peqlab, Erlangen, Germany) and purified RNA was reverse transcribed using the RT.sup.2 First Stand Kit (Qiagen, Hilden, Germany).
[0286] Real-Time PCR.
[0287] RT-PCR reactions were carried out on a BioRad MyiQ (BioRad, Munich, Germany) in a final volume of 25 μL using RT.sup.2 qPCR Primer Assay (for IL-2) and SYBR® Green qPCR Mastermix (both from Qiagen, Hilden, Germany). Each determination was done in duplicate and the housekeeping gene 18s rRNA was used as an internal control. The real-time thermal cycler program consisted of an initial denaturation step at 95° C. for 10 min followed by a two-step cycling program with 40 cycles (95° C., 15 s; and 60° C., 60 s). Results were expressed as relative gene expression of IL-2 and were determined by comparative Ct method. The data were normalized to the Ct value of the internal housekeeping gene 18s rRNA and the relative mRNA level in the untreated group (untreated PHA-L-activated) was used as calibrator.
[0288] Data Analysis and Statistical Analysis.
[0289] For
[0290] FACS graphs and results were prepared using CellQuest Pro Software (BD Biosciences) and are presented as mean+STDEV or SEM. All data pertaining examples 8-12 were statistically evaluated by ANOVA and Dunnet's post hoc-test using SPSS v19.0 (IBM, N.Y., USA).
EXAMPLE 2
Chemical Analysis of Oldenlandia affinis Plant Extract
[0291] The crude extract of the coffee-family plant Oldenlandia affinis was chemically analysed using a rapid peptidomics workflow utilising nano-LC-MS, peptide reconstruct with database identification and MS/MS automated sequence analysis to determine its cyclotide content.
[0292] O. affinis plants were grown and the aerial parts were isolated according to well-known laboratory protocols using overnight extraction with dichloromethane and methanol followed by C.sub.18 solid phase extraction of the aqueous part. This standard procedure commonly yields many grams of crude cyclotide-extract per kilogram of fresh plant leaf weight (Gruber, 2007, Toxicon, 49, 561-575; Gran, 1970, Medd Nor Farm Selsk, 12, 173-180), while the content of various cyclotides depends on the growth conditions (e.g., habitat) of the plants and other environmental factors (Trabi, 2004, J Nat Prod, 67, 806-810; Seydel, 2007, Appl. Microb. Biotechnol., 77, 275-284).
[0293] Generally, amino acid sequencing is only feasible from pure or semi-purified cyclotide fractions. Therefore, an alternative peptidomics approach was used to dissect the cyclotide content from a crude plant extract by combining nanoflow LC-MS and peptide reconstruction (identification by molecular weight) as well as proteolytic digestion, LC-MS/MS and automated database analysis (identification by amino acid sequence) using the recently reported ERA cyclotide database tool (Colgrave, 2010, Biopolymers, 94, 592-601). The crude cyclotide extract was analyzed with various linear gradients on reversed-phase C.sub.18 nano LC coupled online to an electrospray ionization hybrid triple-quadrupole/linear ion-trap (ESI-QqLIT) mass spectrometer, which was operated in enhanced MS mode with scan speeds of 1000 and 4000 amu/sec, respectively. Application of an automated LC-MS reconstruct tool yielded initially a few hundred of peptide masses in the range from 2700-3500 Da (typical MW for cyclotides). The high number likely accounts for some false-positive hits due to the inclusion of low abundant data in the calculation. Hence, the signal-to-noise factor in the algorithm was adjusted and usually between 50-100 reconstructed peptide masses with significant scores above 0.99 were obtained. Representative LC-MS reconstructed data (of at least three independent experiments) are listed in Table 4. A total of 72 peptide masses in the range from 2700-3500 Da were identified. By comparing those peptide masses to the database of cyclotides (CyBase (Wang, 2008, Nucleic Acids Res, 36, D206-210)), 23 known O. affinis cyclotides, 24 peptide masses that correspond to peptides from other cyclotide plant species and 25 new (not previously described) cyclotide masses were identified. LC-MS experiments were further analyzed with manual peptide reconstruction by extracting the doubly- and triply-charged ions of respective cyclotide peaks and by calculation of the average molecular weight (unpublished data). The manual analysis was useful as an internal control to ensure the integrity of the generated automated data.
[0294] In addition to the analysis of O. affinis cyclotides by molecular weight and database comparison, a number of chemical modifications of the crude extract, i.e. reduction and alkylation followed by trypsin and endo-GluC proteolysis, were performed. Due to the structural nature and high stability of cyclotides these chemical modifications are necessary to yield amenable precursor ions for MS/MS sequencing. The modified and digested mixtures were analyzed with a peptidomics workflow utilizing nano LC and peptide sequencing by Information Dependent Acquisition (for further details see the Methods Section). The resulting MS and MS/MS data were used for automated cyclotide identification using the Paragon™ algorithm with a custom-made ERA cyclotide database (a tool that is freely available on the web). Using this cyclotide peptidomics analysis, 14 known cyclotides could be identified by amino acid sequence (see Table 5). In summary, using the above described peptidomics workflow nearly all currently known cyclotides and an even greater number of novel peptide masses corresponding to other known or novel cyclotides (by molecular weight) could be identified in crude cyclotide extract from the plant O. affinis (see Table 1).
[0295] The combination of nano LC-MS/MS and LC-MS reconstruction, as well as automated database searching is a rapid and useful technique for the identification of cyclotides in crude extracts. Compared to an earlier study from Plan et al. (Plan, 2007, ChemBioChem, 8, 1001-1011), which described the first cyclotide fingerprint of O. affinis using classical peptide purification via analytical HPLC and offline MS/MS sequencing, 8 additional known cyclotides have been identified and a list of ˜50 peptide masses has been provided corresponding to cyclotides of which some can be identified by peptide fingerprint analysis in CyBase (the cyclotide database (Wang, 2008, Nucleic Acids Res, 36, D206-210)). This suggests that the number of cyclotides to be found in a single species may be >70 and is, therefore, at least twice the number than earlier anticipated (on average 34 cyclotides per species (Gruber, 2008, Plant Cell, 20, 2471-2483). This, of course, has a huge impact on the determination of the overall number of cyclotides in the plant kingdom and consequently would lead to a necessary revision of the number of novel cyclotides to be discovered in plants.
EXAMPLE 3
Anti-Proliferative Effects of O. affinis Cyclotide Extract
[0296] After completion of the chemical analysis, different concentrations of the crude O. affinis cyclotide extract were tested for its anti-proliferative capacity on activated human primary PBMC (
[0297] The impact of the crude O. affinis cyclotide preparation on the cell division level of activated PBMC was further evaluated. For this purpose, the cells were labeled with the dye carboxyfluorescein diacetate succinimidyl ester (CFSE), which does not influence the viability of the stained cells and is inherited by daughter cells after cell division and each dividing cell consequently loses fluorescent intensity. These data, shown in
EXAMPLE 4
Relative Quantification of Cyclotides and Isolation of Kalata B1
[0298] Since promising anti-proliferative activity of the total cyclotide extract from O. affinis was obtained, the relative amount of the major cyclotides was determined and the main components for biological characterization were further purified. For this purpose the crude cyclotide extract was used for quantitative nano LC-MS analysis, similar as described above. Diluted aliquots of the extract were separated by nano C.sub.18 RP-HPLC coupled online to the mass spectrometer. Eluted peptides were monitored both with absorbance at 214 nm and by molecular weight. The area-under-curve of the major cyclotide peaks in O. affinis was determined by automated integration (and if necessary manual post-processing). The relative quantification analysis of the cyclotide content has been carried out from five independent LC-MS experiments (see Table 6) and a representative O. affinis elution profile, indicating the major cyclotide peaks and their relative abundance (mean±SEM), is shown in
[0299] As presented above, and in agreement with earlier studies (Plan, 2007, ChemBioChem, 8, 1001-1011), the cyclotides kalata B1 and kalata B2 are the main peptide components, accounting for approx. 34% of the overall cyclotide content in O. affinis. Kalata B1 and B2 differ by only five amino acid positions (see
EXAMPLE 5
Anti-Proliferative and Cytotoxic Effects of Kalata B1
[0300] To analyze whether kalata B1 has the capacity to inhibit the proliferation of activated human primary PBMC, the cells were labeled with the fluorescent dye CFSE and analyzed the cell division properties in the presence of the kalata B1 concentrations in the range from 1.8 to 14 μM using flow cytometry. After exposure of PBMC to kalata B1, a dose-dependent decrease of the cell division capacity was observed, as compared to untreated stimulated PBMC controls, as shown in
[0301] To analyze whether the anti-proliferative effect was due to cell damaging, the influence of kalata B1 on the induction of PBMC apoptosis or necrosis was examined (
[0302] The anti-proliferative activity of kalata B1 triggered validation and control experiments to determine the nature of the observed effect. Cytometric-based forward-side-scatter analysis (data not shown) provided solid evidence that the anti-proliferative effect induced by the cyclotide does not cause cell death by either apoptosis or necrosis, but inhibits the growth of the lymphocytes in a cytostatic fashion. Concentrations higher than 14 μM of the peptide are cytotoxic to the cells (data not shown). This was expected since kalata B1 has earlier been reported to cause hemolysis and membrane disruption at concentrations above ˜50 μM (Barry, 2003, Biochemistry, 42, 6688-6695; Henriques, 2011, J Biol Chem, 286, 24231-24241). Therefore, control experiments were performed with the honeybee venom component melittin, a commonly used strong membrane disrupting peptide agent.
[0303] Concentrations were tested, at which cytotoxic effects on human lymphocytes were described in the literature, to ensure that our experimental setup was sensitive enough to detect possible cytotoxic effects of kalata B1 (Pratt, 2005, In Vitro Cell Dev Biol Anim, 41, 349-355) (see
[0304] From these control data, it was concluded that kalata B1, in contrast to melittln, has an anti-proliferative capacity, which is not due to cytotoxic effects and the membrane lysing capacity of kalata B1, as otherwise one would have expected similar observations from the much more potent cytotoxic peptide melittin. The proof of anti-proliferative effects by holding the cells in an “inactive” state at which they are still viable, but aren't able to grow and proliferate without causing cell death in a certain dose range is a crucial precondition to classify a substance as immunosuppressant, because cytotoxicity would cause side effects.
EXAMPLE 6
Test of Cyclotide Mutants/variants in Anti-Proliferative Assays on PBMCs and Isolated T-Lymphocytes
[0305] The anti-proliferative effect of cyclotide mutants/variants was tested according to Example 5. In brief, CFSE-labelled PBMCs, or magnetic-purified CD3+ lymphocytes were stimulated with anti-CD3/28 mAbs, in the presence of medium (ctrl), camptothecin (CPT, 30 μg/mL), cyclosporin A (CsA, 1 μg/mL) or different concentrations of cyclotides (1.8-14 μM) for 72 h. Afterwards the cell proliferation was assessed by analysing cell division using flow cytometric-based histogram analysis. The following peptides (1.8-14 μM) on both PBMCs and CD3-purified T-lymphocytes (n≧2) have been tested:
TABLE-US-00001 Kalata B1: GLPVCGETCVGGTCNTPGCTCSWPVCTRN Kalata B2: GLPVCGETCFGGTCNTPGCSCTWPICTRD D-kalataB2: all-D GLPVCGETCFGGTCNTPGCSCTWPICTRD Kalata T8K; GLPVCGEKCVGGTCNTPGCTCSWPVCTRN Kalata V10A: GLPVCGETCAGGTCNTPGCTCSWPVCTRN Kalata V10K: GLPVCGETCKGGTCNTPGCTCSWPVCTRN Kalata G18K: GLPVCGETCVGGTCNTPKCTCSWPVCTRN Kalata N29K: GLPVCGETCVGGTCNTPGCTCSWPVCTRK Kalata T20K, G1K: KLPVCGETCVGGTCNTPGCKCSWPVCTRN Kalata T20K: GLPVCGETCVGGTCNTPGCKCSWPVCTRN
[0306] The corresponding IC.sub.50 values can be found in Table 2:
TABLE-US-00002 TABLE 2 Comparison of kalata B1 (and other cyclotides) inhibitory effects on PBMC and CD3 purified T-lymphocyte proliferation. Relative activity in other assays IC.sub.50 (μM) (fold difference to kB1) Peptide ±STDEV nematocidal hemolytic insecticidal PBMCs Kalata B1 2.9 ± 1.3.sup.a 1.0 0.7 1.0 Kalata B2 0.2 ± 0.1.sup.c — — — all-D kalata B2 2.3 ± 0.8.sup.c — — — Kalata B1 T8K not active (n.a.).sup.b <0.2 T8A: 0.1 0.2 V10A n.a..sup.b — 0.5 1.1 V10K n.a..sup.b <0.2 — — G18K 4.4 ± 0.5.sup.b 2.4 G18A: 0.6 1.2 N29K 3.2 ± 0.6.sup.b 7.0/3.8 N29A: 0.5 1.0 T20K, G1K 1.9 ± 0.1*.sup.b 6.5/6.8 — — (cytotoxic) T20K 1.9 ± 0.6.sup.c 3.0/2.6 — — MCo59 n.a..sup.b — — — MCo-CC1 n.a..sup.b — — — MCo-CC2 n.a..sup.b — — — CD3 purified lymphocytes Kalata B1 2.4 ± 0.5.sup.d — — — Kalata B2 0.6 ± 0.02.sup.d — — — all-D kalata B2 2.9 ± 0.4.sup.d — — — G18K 3.2 ± 1.8.sup.c — — — N29K 2.1 ± 0.9.sup.c — — — T20K, G1K 1.1 ± 0.7.sup.c — — — (cytotoxic) T20K 2.7 ± 0.6.sup.d — — — *this compound is cytotoxic at 14 μM; all data have been normalized and analyzed with non-linear regression (fixed slope) using Graph Pad, .sup.an = 7, .sup.bn = 4, .sup.cn = 3, .sup.dn = 2; peptides other than kalata B1, have been supplied by David Craik (Institute for Molecular Bioscience, Australia).
EXAMPLE 7
In Vivo Activity in EAE Mouse Model of MS
[0307] The in vivo activity of cyclotides in the EAE mouse model of MS were tested, as described previously (Okuda J Interferon Cytokine Res 18, 1998, 415-421). The ability of mice to recover from motor deficit after developing a chronic progressive form of EAE was examined by vaccinating the mice with kalata B1. MOG MS-like disease model in C57BL/6 mice (Bernard J Mol Med 75, 1997, 77-88) was used, where adult female C57BL/6 (10-12 weeks old) mice were vaccinated with three successive subcutaneous (sc) injections of cyclotides (200 mg each time) in Incomplete Freund's adjuvant (IFA) at weekly intervals before EAE was induced with MOG.sub.35-55. Control mice were similarly treated but received PBS in IFA. Animals were assessed daily for clinical signs of EAE for a period of 43 days.
[0308] Vaccination with kalata B1 resulted in a reduction in the incidence and severity of EAE (
[0309] The influence of kalata B1 vaccination on the formation of CNS inflammatory and demyelinating lesions was examined by histological studies of fixed tissue using haemotoxylin/eosin, Luxol fast blue (LFB) and Bielshowsky silver staining. The CNS of all mice treated with PBS showed extensive inflammatory lesions, characterized by mononuclear inflammatory cells, which were particularly florid in the cerebellum and spinal cord (
[0310] The capacity of spleen cells to proliferate in response to the encephalitogen MOG.sub.35-55 to determine whether the suppressive effect on EAE following vaccination with kalata B1 was associated with a decrease in MOG-specific T cell responses. Furthermore, to address whether this suppression of EAE was antigen specific and/or the result of a defect in the activation or function of T-cells, the same population of splenocytes was stimulated by the polyclonal activators, anti-CD3 and anti-CD28 antibodies.
[0311] Whether the suppression of EAE in mice vaccinated with kalata B1 was associated with a decrease in the production of specific antibodies to MOG was examined. Accordingly, sera from kalata B1 and PBS treated mice were collected at the completion of the experiment (Day 43) and tested for their reactivity to MOG.sub.35-55. As indicated in
[0312] It is well established that the development of EAE is associated with the secretion of proinflammatory cytokines by CNS-antigen specific T cells (Owens Curr Opin Neurol 16, 2003, 259-265). Since the suppression of EAE following kalata B1 vaccination was not associated with a decrease in T cell reactivity to MOG, it was investigated whether MOG-reactive T cells in protected animals may have switched to an anti-inflammatory T cell phenotype. Accordingly, conditioned media generated from in vitro stimulated and non-stimulated spleen cell cultures were assessed in cytokine bead array assays. A total of 15 cytokines were analysed simultaneously, including, IL2, IL3, IL4, IL5, IL6, IL9, IL10, IL12p70, IL13, IFNγ, GM-CSF, KC, MCP1, MIG and TNFα. There were no marked changes in cytokine content in MOG.sub.35-55 or CD3/38-stimulated supernatants between cyclotide and control animal groups (data not shown). In contrast, significantly reduced levels of the chemokine MIG known to play a role in T cell trafficking and TNFα, a pro-inflammatory cytokine known to be involved in the pathogenesis of EAE Nicholson Curr Opin Immunol 8, 1996, 837-842) were demonstrated in non-stimulated spleen cell supernatants generated from animals treated with kalata B1 (
EXAMPLE 8
Influence/Effect of Various Cyclotides on the Expression of IL-2-Alpha-Chain CD25
[0313] Amongst other pathways, T-cell proliferation is determined by binding of the cytokine IL-2 to its cell surface receptor. Therefore the influence of cyclotides on the expression of the IL-2 receptor was tested. The test compounds were T20K, V10A, V10K and T8K and hence PBMCs were treated with these cyclotides, following stimulation with PHA-L in order to determine the expression of the IL-2 surface receptors CD25 after 24 and 48 hours of cultivation, respectively, using FACS analysis (
EXAMPLE 9
Influence of Cyclotides on IL-2 Release and Gene Expression
[0314] To analyze the mechanism of cyclotide-mediated anti-proliferation of T-lymphocytes, their effect on the direct release of IL-2 in PBMCs was determined. The cells were treated with a cyclotide and activated with PHA-L. After 24 h the cells were re-stimulated with PMA and ionomycin and the IL-2 concentration in the supernatant (released IL-2) was measured with an ELISA-based FACS methodology (
[0315] Moreover, supernatants of stimulated T-cells were analyzed for their IL2 release using a human IL-2 ELISA Kit from eBioscience according to the manufacturer's instructions. The color reaction was evaluated at an optical density of 450 nm by the microplate reader Synergy H4 (BioTek) (
[0316] To determine whether cyclotides have an impact at the gene expression level of the, il-2 gene expression (as control we used 18s rRNA) in PBMC cells was investigated by quantitative real-time PCR (
EXAMPLE 10
Influence of Exogenous IL-2 Addition to Cyclotide-Treated PBMCs
[0317] To determine the validity of the significant reduction of IL-2 release after cyclotide treatment, the influence of exogenous addition of IL-2 post treatment was tested. If IL-2 synthesis is reduced by treatment with CsA and cyclotides, one would expect that this effect can be reversed by exogenous addition of IL-2 to the treated cells. Therefore, PBMCs were treated with cyclotides and CsA and the cells were activated with PHA-L. In parallel, the cells were grown with addition of exogenous IL-2 (
EXAMPLE 11
Influence of Cyclotides on the IFN-Gamma or TNF-Alpha Production
[0318] From the results so far it is evident that treatment of activated PBMCs with CsA or cyclotide T20K influences the expression of the IL-2 surface receptor CD25 (
[0319] CsA (23%±1.8) and T20K (23%±10.6) also led to a significant (p≦50.001) reduced TNF-alpha expression as compared to the control (
EXAMPLE 12
Influence of Cyclotides on the Degranulation Activity of Activated PBMCs
[0320] After determining the Influence of cyclotide treatment on the effector function of PBMCs on the basis of measuring IFN-gamma and TNF-alpha cytokine levels, it is of Interest to determine an effect of cyclotides on the degranulation activity. Activation of cytotoxic CD8.sup.+-lymphocytes lead to a release of cytolytic granules, which contain/express lysosomal-associated membrane protein 1 (CD107; LAMP-1). During degranulation, the granule vesicle membranes fuse with the membranes of activated CD8′-lymphocytes and therefore LAMP-1 can be used as a marker protein for the cytotoxic activity of T-lymphocytes, which can be measured with FACS. After 36 h, 42%±21.4 of the CsA- and 49%±16.8 of the T20K-treated cells contain the degranulation marker LAMP-1 as compared to the control (
EXAMPLE 13
Ca.SUP.2+ .Release of Jurkat Cells
[0321] Jurkat cells T-cells were treated as described for
EXAMPLE 14
Effect of Cyclotides on C57BL/6J Mice
Materials
[0322] Seven weeks old female C57BL/6J mice were purchased from the Department for Lab-zoology and -genetics (Himberg, Austria). All experiments were approved according to the European Community rules of animal care with the permission of the Austrian Ministry of Science. T20K and V10K were provided by D. J. Craik, from the University of Queensland, Institute for Molecular Bioscience (Brisbane, Australia). 5-carboxyfluoresceine-N-hydroxysuccinimid was purchased from Sigma-Aldrich (Vienna, Austria).
Immunization
[0323] Mice (n=10/group) were treated on day (−7), 0, 7 with 200 μg/100 μL/mouse T20K solubilized in sterile PBS intraperitoneally (i.p.), as indicated in the figure. On day 0 they were immunized subcutaneously with myelin oligodendrocyte glycoprotein (MOG.sub.35-55, 1 mg/mL) and complete Freud's adjuvant (CFA, 10 mg/mL) mixed at equal parts. Therefore 70 μL were injected into the left and right flank. Additionally mice received 100 μL pertussis toxin (2 μg/mL) i.p. on day 0 and again on day 2. Beginning at day 10 mice were scored every second day. Weight was also measured at day (−7), 0, 7 and on the same day during scoring. Mice were sacrificed on day 24 after reaching high scores.
Spleenocyte Isolation and Stimulation
[0324] Spleens of sacrificed mice were taken and transferred into a 6 cm Petri Dish with 5 mL sterile PBS. To receive a spleenocyte suspension, spleens were meshed and filtered through 70 μm nylon sieve. Cells were centrifuged at 1200 rpm for 5 minutes and resuspended in RPMI 1640 media supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL). Spleenocytes were cultivated at a concentration of 3×10.sup.6/mL in a 48-well flat-bottom plate (500 μL/well). Cells were stimulated with 30 μg/mL MOG.sub.35-55 or left untreated and incubated at 37° C. for three days. Supernatants and cells were taken stored at −20° C. until further experiments. Cells isolated from the naïve mouse group were additionally cultivated in a 96-well flat-bottom plate (100 μL/well) and stimulated with T20K (4 μM), T20K+MOG, T20K (12 h), V10K (4 μM, 12 h), CsA (5 μg/mL; 12 h), MOG (12 h) or left untreated. After 12 hours MOG or T20K was added to appropriate wells. Supernatants were stored after 24 hours and after 48 hours at −20° C.
Enzyme Linked Immunosorbent Assay
[0325] Supernatants of stimulated spleenocytes were analyzed for their IL2, IL17, INFγ, IL4 and IL22 cytokine release using anti-mouse antibodies for ELISA from eBioscience according to the manufacturer's instructions. The color reaction was evaluated at an optical density of 450 nm by the microplate reader Synergy H4 (BioTek).
SDS-PAGE and Western Blotting for NFAT1c
[0326] Human T-cells provided by CCRI from A. Dohnal, PhD were stimulated according to the protocol for IL2 release described above. Stimulated cells were resuspended in TBS and mixed at equal parts with sample buffer and heated for five minutes at 95° C. A sodium dodecyl sulfate polyacrylamide gel was prepared to separate the proteins achieved from the lysed T-cells. After electrophoresis proteins were transferred from the gel to a membrane. After blocking the membrane with BSA 3% in TBST over night at 4° C., the first antibody mouse anti-NFATc1 was incubated for 2 hours at room temperature. After five times washing with TBST 0.1% Tween, the membrane was incubated with the second antibody anti-mouse IgG HRP for one hour at room temperature. The membrane was dried and treated with West Pico or West Femto Super Signal Solution according to the manufactures protocol to evaluate the chemo-luminescence signal.
EXAMPLE 15
Cell Permeability of T20K Cyclotides (Chemical Labelling and Microscopy)
[0327] 1.5 mg of T20K was dissolved in 1.5 ml of 100 mM sodium carbonate buffer of pH 8,8. 5-carboxyfluoresceine-N-hydroxysuccinimid ester (5-CFSE) was added in 10 fold excess as solid compound (2.5 mg) The reaction was allowed to proceed for 120 min at room temperature. Afterwards the reaction mixture was heated to 50° C. for 30 min to complete the hydrolysis of the N-hydroxysuccinimid ester (NHS). Purification was performed using semi-preparative chromatography, applying a Kromasil RP column 250×10 ID, 5 μm 100 Å. Eluent A was ddH.sub.2O/TFA 99.9/0.1% (v/v), eluent B was AcN/H.sub.2O/TFA 90/10/0.08% (v/v/v). The linear gradient from 5% eluent B to 80% eluent B in 50 min was used. Maldi-TOF-MS analysis of the collected fractions yielded a mass of 3276.3 Da in one of the fractions. The mass peak of 3276.3 Da were identified as the mono-derivatized species of T20K with 5-carboxyfluoresceine with a mass shift of 357 Da. Human T-cells and Jurkat cells were incubated with a 4 μM solution of the T20K derivative in RPMI 1640 media supplemented with the additives described above for 20 min. The fluorescence microscope was from Zeiss LSM 510 confocal microscope. The excitation wavelength was 488 nm and emission wavelength 520 nm.
EXAMPLE 16
[0328] The present invention refers to the following supplemental tables:
TABLE-US-00003 TABLE 1 Cyclotides from O. affinis extract identified by nano LC-MS and MS/MS Theoretical MW (avg.) MW (mono.) MW Δ MW Cyclotide.sup.1 Da.sup.2 Da.sup.2 Score.sup.3 Evidence.sup.4 Da.sup.5 Da.sup.6 kalata B1 2892.85 2890.39 1 ICP 2892.33 0.52 kalata B2 2956.14 2953.74 1 ICS 2955.38 0.76 kalata B3 3083.31 3080.64 1 ICS 3082.48 0.83 kalata B4 2893.24 2890.56 1 IS 2893.31 0.07 kalata B5 — — — P 3061.59 — kalata B6 3029.96 3027.66 0.9999 IS 3029.42 0.54 kalata B7 3072.26 3069.74 0.9998 IS 3071.59 0.67 kalata B8 3284.34 3281.75 1 ICS 3283.79 0.55 kalata B9 — — — P 3272.72 — kalata B9 lin — — — P 3290.74 — kalata B10 3030.21 3027.53 1 ICS 3030.41 0.20 kalata B10 lin 3048.54 3046.50 1 ICS 3048.43 0.11 kalata B11 2884.48 2881.44 0.9999 I 2884.26 0.22 kalata B12 — — — P 2880.27 — kalata B13 3036.06 3033.58 1 IC 3036.46 0.40 kalata B14 3023.74 3021.17 0.9987 I 3022.43 1.31 kalata B15 2977.00 2974.56 1 ICS 2976.40 0.60 kalata B18 3147.33 3145.02 0.9977 I 3145.67 1.66 kalata S 2878.81 2875.93 0.9993 I 2878.30 0.51 Oak6 cyclotide 1 3035.87 3033.49 1 IC 3035.47 0.40 [G-A] kalata B1.sup.7 2906.47 2904.75 0.9995 I 2906.35 0.12 kalata b1-1 2724.12 2722.28 1 IC 2724.18 0.06 [L2A] kalata B1 2851.88 2849.54 1 IC 2850.25 1.63 Ac-[desGly]-KB1-Am 2854.31 2851.68 0.9996 I 2853.30 1.01 acyclic kalata B1 2911.32 2908.36 1 IC 2910.35 0.97 Oak6 cyclotide 2 3093.29 3090.61 1 IC 3092.56 0.73 .sup.1Identification by LC-MS reconstruct of at least 3 representative LC-MS experiments (±1 Da, 20-70 min, EMS 1000 2 scans) or identification by digest (trypsin or endo-GluC), nano LC-MS/MS and database search (ERA); .sup.2Observed molecular weight; .sup.3Score indicating the quality of LC-MS reconstructed peptide MW (≦1.0); .sup.4Evidence for identified cyclotides, I = isotope pattern, C = charge pattern, S = full sequence, P = partial sequence or sequence tag; .sup.5Data taken from CyBase (Wang, 2008, Nucleic Acids Res, 36, D206-210); .sup.6Δ MW determined to average molecular weight; .sup.7 amino acid position (G-A replacement) not specified
TABLE-US-00004 TABLE 3 Comparison of kalata B1 (and other cyclotides) inhibitory effects IC50 values in various cellular test systems. Assay system Cells IC.sub.50 (μM) Reference kalata B1 Anti-proliferative activity human peripheral blood 3.9 ± 0.5 Gründemann et al., mononuclear cells 2012 Nematocidal activity H. contortus nematodes 2.7 Huang et al., 2010 T. colibriformus nematodes 4.5 Huang et al., 2010 Cytotoxicity human T-lymphoblast cells 3.5 Daly et al., 2004 other cyclotides* Cytotoxicity human lymphoma cell line 0.6-6 Svangard et al., 2004 (U-937) 0.3-7 Lindholm et al., 2002 Cytotoxicity human myeloma cell line 1-4 Svangard et al., 2004 (RPMI-8226/s) 0.1-6 Lindholm et al., 2002 *Activity was reported of various cyclotides (varv A, varv E, varv F, vitri A, cycloviolacin O2) from Viola arvensis, V. odorata and V. tricolor
TABLE-US-00005 TABLE 4 LC-MS reconstruct of O. affinis cyclotides. Raw (labelled) data of LC-MS reconstruct of O. affinis extracts as analysed by nano LC-MS. LC-MS reconstruct, EMS 1000 Da/sec Reconstruct 2700-3500 Da, signal-to-noise: 4, 25, 50; combined datasets from at least 3 independent LC-MS runs CyBase comparison: MW +− 1 Da *= other cylotide detected (not Oaffinis) **= MW +/− 2 Da Theoretical Mass Da Mass Δ Mass No. cyclotide Da (avg.) (mono.) Score Evidence Da (avg.) Da 1 new 2706.63 2704.37 0.9997 I 2 new 2723.22 2721.27 1 I 3 kalata b1-1 2724.12 2722.28 1 IC 2724.18 0.0559 4 new 2821.78 2819.36 1 IC 5 new 2822.30 2820.55 0.9995 I 6 new 2833.30 2831.41 1 I 7 [L2A] kalata B1 2851.88 2849.54 1 IC 2850.25 1.6322 8 Ac-[desGly]-KB1- 2854.31 2851.68 0.9996 I 2853.3 1.0072 Am 9 new 2873.73 2871.13 0.9996 I 10 kalata S 2878.81 2875.93 0.9993 I 2878.30 0.5141 11 new 2879.82 2877.46 1 IC 12 kalata B12** 2882.30 2880.83 0.9969 I 2880.27 2.0256 13 kalata B11 2884.48 2881.44 0.9999 I 2884.26 0.2236 14 new 2891.45 2888.50 1 IC 15 kalata B1 2892.85 2890.39 1 IC 2892.33 0.5228 16 kalata B4 2893.24 2890.56 1 I 2893.31 0.0718 17 new* 2896.70 2894.45 0.9999 I 18 new 2897.11 2894.57 1 IC 19 [G-A] kalata B1 2906.47 2904.75 0.9995 I 2906.35 0.1191 20 new 2909.53 2906.90 1 IC 21 acyclic kalata B1 2911.32 2908.36 1 IC 2910.35 0.9675 22 new* 2912.90 2911.43 0.9999 I 23 new* 2922.95 2920.48 1 IC 24 new* 2925.32 2922.40 1 IC 25 new* 2926.80 2923.70 1 IC 26 new 2927.30 2924.66 1 IC 27 new 2937.91 2935.50 0.9998 I 28 new 2942.99 2940.48 1 IC 29 kalata B2 2956.14 2953.74 1 IC 2955.38 0.7637 30 new* 2959.95 2957.56 1 IC 31 new 2960.36 2958.24 0.9996 I 32 new 2969.25 2968.13 0.9997 I 33 new* 2971.44 2969.50 1 IC 34 new 2973.80 2970.55 1 IC 35 new 2974.14 2971.51 1 IC 36 new* 2975.38 2973.49 1 IC 37 kalata B15 2977.00 2974.56 1 IC 2976.40 0.602 38 new* 2986.57 2983.80 1 I 39 new 2988.28 2985.60 1 IC 40 new 2990.37 2987.51 1 IC 41 new 2994.11 2991.86 1 IC 42 new* 3006.25 3003.50 1 IC 43 new* 3010.97 3008.88 1 IC 44 kalata B14 3023.74 3021.17 0.9987 I 3022.43 1.3147 45 new* 3028.61 3025.92 0.9998 I 46 kalata B6 3029.96 3027.66 0.9999 I 3029.42 0.5381 47 kalata B10 3030.21 3027.53 1 IC 3030.41 0.2028 48 Oak6 cyclotide 1 3035.87 3033.49 1 IC 3035.47 0.398 49 kalata B13 3036.06 3033.58 1 IC 3036.46 0.4018 50 new 3039.91 3037.45 1 IC 51 new 3040.05 3036.62 1 IC 52 new* 3045.78 3043.50 1 IC 53 new* 3046.32 3044.95 1 IC 54 new 3047.97 3046.60 0.9999 I 55 kalata B10 in 3048.54 3046.50 1 IC 3048.43 0.1091 56 new* 3051.82 3048.48 1 IC 57 new* 3052.72 3049.57 1 IC 58 new* 3065.79 3063.36 0.9997 I 59 kalata B7 3072.26 3069.74 0.9998 I 3071.59 0.67 60 new* 3073.89 3072.70 0.9999 I 61 kalata B3 3083.31 3080.64 1 IC 3082.48 0.8309 62 new* 3087.22 3084.61 1 IC 63 new 3089.27 3086.96 0.9997 I 64 new 3091.00 3089.03 0.9997 I 65 Oak6 cyclotide 2 3093.29 3090.61 1 IC 3092.56 0.7328 66 new* 3097.63 3094.57 1 IC 67 new* 3099.85 3096.60 1 IC 68 kalata B18** 3147.33 3145.02 0.9977 I 3145.67 1.6615 69 new* 3266.81 3264.99 0.9997 I 70 kalata B8 3284.34 3281.75 1 IC 3283.79 0.5453 71 new* 3300.96 1 C 72 new 3446.88 3444.98 0.9998 I Total: 72 New: 25 New*: 24
TABLE-US-00006 TABLE 5 O. affinis database search results following digests and LC-MS/MS analysis. Protein Pilot ™ database search results of the cyclotide LC-MS/MS analysis. Peptides N Unused Total % Cov % Cov (50) % Cov (95) Accession Name (95%) trypsin digest 1 6.43 6.43 100.00 85.48 82.26 cb|P85175 kalata B8/1-31|cybaseid = 168 organism = 4 Oldenlandia affinis 2 6.00 6.00 100.00 51.72 51.72 cb|P58457 kalata B7/1-29|cybaseid = 26 organism = 6 Oldenlandia affinis 3 5.91 5.91 100.00 98.28 96.55 cb|P58454 kalata B2/1-29|cybaseid = 4 organism = 4 Oldenlandia affinis 4 2.75 2.75 100.00 98.28 72.41 cb|P83938 kalata B4/1-29|cybaseid = 30 organism = 2 Oldenlandia affinis 5 2.63 2.63 93.33 88.33 88.33 cb|P58456 kalata B5/1-30|cybaseid = 59 organism = 5 Oldenlandia affinis 6 2.00 3.75 100.00 98.28 72.41 cb|P85133 kalata B15/1-29|cybaseid = 253 organism = 3 Oldenlandia affinis 7 2.00 2.00 100.00 50.00 50.00 cb|P85128 kalata B10/1-30|cybaseid = 246 organism = 1 Oldenlandia affinis 7 0.00 2.00 100.00 50.00 50.00 cb|247 kalata B10 linear/1-30|cybaseid = 247 organism = 1 Oldenlandia affinis 8 2.00 2.00 85.48 85.48 43.55 cb|P85127 kalata B9/1-31|cybaseid = 244 organism = 1 Oldenlandia affinis 8 0.00 2.00 85.48 85.48 43.55 cb|245 kalata B9 linear/1-31|cybaseid = 245 organism = 1 Oldenlandia affinis 9 2.00 2.00 98.21 50.00 50.00 cb|P85130 kalata B12/1-28|cybaseid = 250 organism = 2 Oldenlandia affinis 10 1.06 2.00 100.00 50.00 50.00 cb|P58455-b3 kalata B6/1-30|cybaseid = 24 organism = 1 Oldenlandia affinis 10 0.00 2.00 100.00 50.00 50.00 cb|247 kalata B10 linear/1-30|cybaseid = 247 organism = 1 Oldenlandia affinis 11 0.63 2.01 98.28 98.28 72.41 cb|P56254 kalata B1/1-29|cybaseid = 1 organism = 4 Viola odorata; Oldenlandia affinis; Viola baoshanensis; Viola yedoensis 12 0.20 0.20 100.00 61.67 0.00 cb|P58455-b6 kalata B3/1-30|cybaseid = 25 organism = 0 Oldenlandia affinis Endo GluC digest 1 0.88 0.88 100 87.92999983 0 cb|P58454 kalata B2/1-29|cybaseid = 4 organism = 0 Oldenlandia affinis 2 0.52 0.52 100 50 50 cb|P58457 kalata B7/1-29|cybaseid = 26 organism = 1 Oldenlandia affinis 3 0.21 0.21 100 25 0 cb|P58455-b6 kalata B3/1-30|cybaseid = 25 organism = 0 Oldenlandia affinis
TABLE-US-00007 TABLE 6 Cyclotide quantification data. Data of five independent experiments of cyclotide quantification. Identified MW calculated Δ MW MW LC-MS reconstruct Δ MW MW theoretical cyclotide (Da) SEM (Da) (Da) SEM (Da) (Da) kalata B8 3283.91 0.15 0.12 3284.24 0.19 0.45 3283.79 kalata B7 3071.83 0.19 0.24 3072.27 0.17 0.68 3071.59 kalata B1 2892.27 0.27 0.06 2892.98 0.21 0.65 2892.33 kalata B6 3029.60 0.28 0.18 3029.84 0.12 0.42 3029.42 kalata B13 3035.56 0.20 0.90 3035.89 0.12 0.57 3036.46 kalata B2 2955.76 0.12 0.38 2955.90 0.09 0.52 2955.38 kalata B3 3082.72 0.21 0.24 3083.04 0.08 0.55 3082.48 Identified Area Height cyclotide RT (min) SEM (mAU * min) SEM (mAU) SEM Rel. Area % SEM kalata B8 29.95 0.01 20.55 0.57 28.01 0.72 5.6 0.2 kalata B7 37.54 0.04 7.89 0.22 41.43 0.21 2.2 0.1 kalata B1 45.48 0.02 50.81 0.84 96.96 0.61 13.9 0.2 kalata B6 46.50 0.03 29.63 0.46 46.88 0.38 8.1 0.1 kalata B13 50.32 0.03 14.88 1.16 28.75 0.38 4.1 0.3 kalata B2 51.82 0.03 72.86 3.17 103.65 0.86 20.0 0.6 kalata B3 52.59 0.07 11.00 1.40 23.02 1.27 3.0 0.4 *n = 5. HPLC quantification (area under curve) of five independent experiments **MW average mass ***MW calculated from +2 or +3 ion
[0329] The present invention refers to the following nucleotide and amino acid sequences:
TABLE-US-00008 SEQ ID No. 1: Amino acid sequence of Kalata B1: GLPVCGETCVGGTCNTPGCTCSWPVCTRN SEQ ID No. 2: Amino acid sequence of Kalata B2: GLPVCGETCFGGTCNTPGCSCTWPICTRD SEQ ID No. 3: Amino acid sequence of D-Kalata B2: all-D GLPVCGETCFGGTCNTPGCSCTWPICTRD SEQ ID No. 4: Amino acid sequence of Kalata G18K: GLPVCGETCVGGTCNTPKCTCSWPVCTRN SEQ ID No. 5: Amino acid sequence of Kalata N29K: GLPVCGETCVGGTCNTPGCTCSWPVCTRK SEQ ID No. 6: Amino acid sequence of Kalata T20K, G1K: KLPVCGETCVGGTCNTPGCKCSWPVCTRN SEQ ID No. 7: Amino acid sequence of Kalata T20K: GLPVCGETCVGGTCNTPGCKCSWPVCTRN SEQ ID No. 8: Amino acid sequence of Kalata T8K: GLPVCGEKCVGGTCNTPGCTCSWPVCTRN SEQ ID No. 9: Amino acid sequence of Kalata V10A: GLPVCGETCAGGTCNTPGCTCSWPVCTRN SEQ ID No. 10: Amino acid sequence of Kalata V10K: GLPVCGETCKGGTCNTPGCTCSWPVCTRN SEQ ID No. 11: Nucleotide sequence encoding Kalata B1: GGACTTCCAGTATGCGGTGAGACTTGTGTTGGGGGAACTTGCAACACTCC AGGCTGCACTTGCTCCTGGCCTGTTTGCACACGCAAT SEQ ID No. 12: Nucleotide sequence encoding Kalata B2: GGTCTTCCAGTATGCGGCGAGACTTGCTTTGGGGGAACTTGCAACACTCC AGGCTGCTCTTGCACCTGGCCTATCTGCACACGCGAT SEQ ID No. 13: Amino acid sequence of the Kalata B1 precursor protein. The mature Kalata B1 domain is underlined. P56254, Kalata-B1, Oldenlandia affinis MAKFTVCLLLCLLLAAFVGAFGSELSDSHKTTLVNEIAEKMLQRKILDGV EATLVTDVAEKMFLRKMKAEAKTSETADQVFLKQLQLKGLPVCGETCVGG TCNTPGCTCSWPVCTRNGLPSLAA SEQ ID No. 14: Amino acid sequence of the Kalata B2 precursor protein. The three mature Kalata B2 domains are underlined. P58454, Kalata-B2, Oldenlandia affinis MAKFTNCLVLSLLLAAFVGAFGAEFSEADKATLVNDIAENIQKEILGEVK TSETVLTMFLKEMQLKGLPVCGETCFGGTCNTPGCSCTWPICTRDSLPMR AGGKTSETTLHMFLKEMQLKGLPVCGETCFGGTCNTPGCSCTWPICTRDS LPMSAGGKTSETTLHMELKEMQLKGLPVCGETCFGGTCNTPGCSCTWPIC TRDSLPLVAA SEQ ID No. 15: Nucleotide sequence encoding the Kalata B1 pre- cursor protein. The nucleotide sequence corresponding to the mature Kalata B1 domain is underlined. >gi|15667740|gb|AF393825.1|Oldenlandia affinis kalata B1 precursor, mRNA, complete cds GGCACCAGCACTTTCTTAAAATTTACTGCTTTTTCTTATTTCTTGTTCTG TGCTTGCTTCTTCCATGGCTAAGTTCACCGTCTGTCTCCTCCTGTGCTTG CTTCTTGCAGCATTTGTTGGGCCGTTTGGATCTGAGCTTTCTGACTCCCA CAAGACGACCTTGGTCAATGAAATCGCTGAGAAGATGCTACAAAGAAAGA TATTGGATGGAGTGGAAGCTACTTTGGTCACTGATGTCCCCGAGAAGATG TTCCTAAGAAAGATGAAGGCTGAAGCGAAAACTTCTGAAACCGCCGATCA GGTGTTCCTGAAACAGTTGCAGCTCAAAGGACTTCCACTATGCGGTGAGA CTTGTGTTGGCGCAACTTGCAACACTCCAGGCTCCACTTGCTCCTGGCCT GTTTGCACACGCAATGGCCTTCCTAGTTTGGCCGCATAATTTGCTTGATC AAACTGCAAAAATGAATGAGAAGGCCGACACCAATAAAGCTATCAATGTA GTTGGTCCCTGTACTTAATTTGGTTGGCTCCAAACCATGTGTGCTGCTCT TGTTTTTGTTTTTTCTTTTTTCTTCTCTCTTTCGGGCACTCTTCAGGACA TGAAGTGATGATCAGTACTCTTTGCTATCATGTTTTCTGTGCACACCTTC TATTGTAGGTGTTGTTGTGATGTTGATGCCCAATTGGATAACTGTTGTCG TTGTTAAAAAAAAAAAAAA SEQ ID No. 16: Nucleotide sequence of encoding the Kalata B2 pre- cursor protein. The nucleotide sequences corresponding to the three mature Kalata B2 domais are underlined. >gi|15667746|gb|AF393828.1|Oldenlandia affinis kalata B2 precursor, mRNA, complete cds GGCACCAGATACAACCCCTTTCTTATAATTTATTGCTTTTCTTATTCCTT GAAAAAGGAGAAATAATATTGGATCTTCCATGGCTAAGTTCACCAACTGT CTCGTCCTGAGCTTGCTTCTAGCAGCATTTGTTGGGGCTTTCGGAGCTGA GTTTTCTGAAGCCGACAAGGCCACCTTGGTCAATGATATCGCTGAGAATA TCCAAAAAGAGATACTGGGCGAAGTGAAGACTTCTGAAACCGTCCTTACG ATGTTCCTGAAAGAGATGCAGCTCAAAGGTCTTCCAGTATGCGGCGAGAC TTGCTTTGGGGGAACTTGCAACACTCCAGGCTGCTCTTGCACCTGGCCTA TCTGCACACGCGATAGCCTTCCTATGAGGGCTGGAGGAAAAACATCTGAA ACCACCCTTCATATGTTCCTGAAAGAGATGCAGCTCAAGGGTCTTCCAGT TTGCCGCGAGACTTGCTTTGGGGGAACTTGCAACACTCCAGGCTGCTCGT GCACCTGGCCTATCTGCACACGCGATAGCCTTCCTATGAGTGCTGGAGGA AAAACATCTGAAACCACCCTTCATATGTTCCTGAAAGAGATGCAGCTCAA GGGTCTTCCAGTTTGCGGCGAGACTTGCTTTGGGGGAACTTGCAACACTC CAGGCTGCTCGTGCACCTGGCCTATATGCACACGTGATAGCCTTCCTCTT GTGGCTGCATAATTTGCTTCATCAAACTGCAAAATGAATAAGAAGGGACA CTAAATTAGCTATGAATTTTGTTGGCCCTTGTGTCTGGTAATTTGGTTCC CGCCAAATTAACCATATGTATGCATTGCTCCTTTTTTCTTTCTTTTTTTT CCCCCTCATTTGGGCACTCTTCATTACATGAAGAGATCATGACGCTTTGT TACTCTGAGCACCCCCTGTTGGTGTTGTTCACATGTTGATGCCCATGTTG GAATAAACTCTTGTTTTTGTTACCAAAAAAAAAAAAAAAAAAA SEQ ID No. 17: Consensus amino acid sequence of active Cyclotides (Xxx.sub.1 is any amino acid, non-natural amino acid or peptidomimetic; Xxx.sub.2 is any amino acid, non- natural amino acid or peptidomimetic but not Lys; and Xxx.sub.3 is any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys): Xxx.sub.1-Leu-Pro-Val-Cys-Gly-Glu-Xxx.sub.2-Cys-Xxx.sub.3-Gly- Gly-Thr-Cys-Asn-Thr-Pro-Xxx.sub.1-Cys-Xxx.sub.1-Cys-Xxx.sub.1- Trp-Pro-Xxx.sub.1-Cys-Thr-Arg-Xxx.sub.1
FURTHER REFERENCES
[0330] 1. Ewing Immunol Cell Biol 76, 47-54 (1998). [0331] 2. Bernard Curr Opin Immunol 4, 760-765 (1992). [0332] 3. Onuki Microsc Res Tech 52, 731-739 (2001). [0333] 4. Ayers Neurochem Int 45, 409-419 (2004). [0334] 5. Bemard J Mol Med 75, 77-88 (1997). [0335] 6. Dubois J Neurol Neurosurg Psychiatry 74, 946-949 (2003). [0336] 7. Krause Febs J 274, 86-95 (2007). [0337] 8. Reiss Platelets 17, 153-157 (2006). [0338] 9. Zhou J Virol 81, 7517-7528 (2007). [0339] 10. dos Santos J Neuroimmunol 162, 122-129 (2005). [0340] 11. Martin Nat Biotechnol 21, 71-76 (2003). [0341] 12. Craik J Mol Biol 294, 1327-1336 (1999). [0342] 13. Craik Science 311, 1563-1564 (2006). [0343] 14. Craik Biopolymers 84, 250-266 (2006). [0344] 15. Rosengren J Biol Chem 278, 8606-8616 (2003). [0345] 16. Simonsen, J Biol Chem 283, 9805-9813 (2008). [0346] 17. Cemazar Internat. J. of Peptide Research and Therapeutics 12, 253-260 (2006). [0347] 18. Colgrave Biochemistry 43, 5965-5975 (2004). [0348] 19. Okuda J Interferon Cytokine Res 18, 415-421 (1998). [0349] 20. Schnolzer Int J Pept Protein Res 40, 180-193 (1992). [0350] 21. Dawson Science 266, 776-779 (1994). [0351] 22. Albouz-Abo Eur J Biochem 246, 59-70 (1997). [0352] 23. Hvas Scand J Immunol 46, 195-203 (1997). [0353] 24. Johns Mol Immunol 34, 33-38 (1997). [0354] 25. Menon J Neurochem 69, 214-222 (1997). [0355] 26. Liu Nat Med 4, 78-83 (1998). [0356] 27. Slavin Autoimmunity 28, 109-120 (1998). [0357] 28. Ichikawa Int Immunol 8, 1667-1674 (1996). [0358] 29. Ichikawa J Immunol 157, 919-926 (1996). [0359] 30. Bernard Clin Exp Immunol 52, 98-106 (1983). [0360] 31. Pedersen J Neuroimmunol 5, 251-259 (1983). [0361] 32. Bettadapura J Neurochem 70, 1593-1599 (1998). [0362] 33. Okuda J Neuroimmunol 131, 115-125 (2002). [0363] 34. Ichikawa Cell Immunol 191, 97-104 (1999). [0364] 35. Daly Biochemistry 38, 10606-10614 (1999). [0365] 36. Cemazar J Biol Chem 281, 8224-8232 (2006). [0366] 37. Owens Curr Opin Neurol 16, 259-265 (2003). [0367] 38. Nicholson Curr Opin Immunol 8, 837-842 (1996). [0368] 39. Gonzalo J Immunol 166, 1-5 (2001). [0369] 40. Henry Immunol Today 20, 285-288 (1999). [0370] 41. Ihle Nature 377, 591-594 (1995). [0371] 42. Janeway Cell 76, 275-285 (1994). [0372] 42. Minami Annu. Rev. Immunol 11, 245-268 (1993). [0373] 43. Svanborg Curr Opin Microbiol 2, 99-105 (1999). [0374] 44. DeVries Semin Immunol 11, 95-104 (1999). [0375] 45. Larsson Inflammation 23, 217-230 (1999). [0376] 46. Cerdan Res Immunol 146, 164-168 (1995). [0377] 47. Jain Cure Opin Immunol 7, 333-342 (1995). [0378] 48. Romagnani Inflamm Bowel Dis 5, 285-294 (1999). [0379] 49. Orlinick Cell Signal 10, 543-551 (1998). [0380] 50. Romanic Lab Invest 76, 11-23 (1997). [0381] 51. Hom Immunobiology 202, 151-167 (2000). [0382] 52. Callan J Clin Invest 106, 1251-1261 (2000). [0383] 53. Grundemann J Nat Prod 75, 167-174 (2012). [0384] 54. Betts J Immunol Methods 281, 65-78 (2003). [0385] 55. Abraham Curr Opin Immunol 10, 330-336 (1998). [0386] 56. Ohta J Immunol 183, 5487-5493 (2009).